Tumor necrosis factor (TNF) is a protein having a molecular weight of about 39,000. It has been cloned from a number of species, and has been expressed in several expression systems. Additionally, muteins of recombinant TNF have been constructed which lack amino acids from the N-terminus of the molecule, or which lack, or have greatly reduced cysteine content. The biological effects of TNF were first described by Carswel et al., (PNAS (U.S.A.) (1975) 72:3666) as a factor present in serum that was induced by endotoxin, and which caused necrosis of certain types of tumors. More recently, recombinantly produced human TNF has also been shown to be an effective anti-cancer agent (Pannica et al., (Nature (London) (1984) 312:724-729), Shirai et al., (Nature (London) (1985) 313:803), Wang et al., (Science (1985), 228:149). TNF has also been reported to inhibit a variety of bacterial or viral infections, either alone or in combination with other lymphokines or cytokines.
Other activities ascribable to TNF include activation of polymorphonuclear leukocytes (Shalaby et al. Journal of Immunology (1985), 135:2069), stimulation of bone resorption and inhibition of bone formation (Bertolini et al., Nature, 319:516), inhibition of lipoprotein lipase (Beutler et al., Nature, 316:552 (1985)), and stimulation of collagenase and prostaglandin E.sub.2 production, (Dayer et al., Journal Experimental Medicine, 162:2163 (1985)). TNF is also thought to stimulate fibroblast growth, (Sugarman et al., Science, 230:943 (1985)) and induce interleukin-1, (Nawroth et al., Journal Experimental Medicine, 163:1363 (1986)).
Despite the enormous clinical potential of TNF, suitable prophylactic or therapeutic formulations of the molecule have not been described. In this regard, it is desirable to have formulations that stabilize or maintain the biological activity of TNF, as well as prevent or retard the formation of aggregates, or oligomers, of TNF. The two phenomena are generally unrelated in that biological activity may be independent of, and not affected by aggregation. Aggregated biomolecules, however, are known to have the undesirable property of increased immunogenicity. Bach, J-F. Immunologic Tolerance in Immunology Immunology 2nd ed. chap. 20., pages 575-590 (Ed J-F Bach, R. S. Schmartz, 1982).
Additionally, it is further desirable to develop TNF formulations that are free of particulate matter, as visual clarity in the clinical setting is often considered by the physican as confirmation that the formulation meets U.S.P criteria. Formulations administered by injection should be free of visible particulate matter such that they should not contain more than 1000 or 10,000 particles having sizes of 25 .mu.m and 10 .mu.m, respectively. Moreover, for obvious economic reasons, it is particularly important to develop a formulation that is free of particulate matter for the shelf life of the formulation.
Lastly, it is apparent that it is desirable to have formulations that are relatively temperature insensitive, and display all the properties described above over a significant temperature range.
Formulations that stabilize TNF activity are shown in European Patent Application 83301740.3, inventors Sakamoto et al., and in European Patent Application 85106915.3, inventors Sakamoto et al. The former shows a method for stabilizing TNF, either in solution or in solid form using a combination of albumin, gelatin, globulins, protamines, and a salt thereof. It is important to note that while the activity of TNF is enhanced, this method does not reduce TNF oligomer formation. European Patent Application 85106915.3 similarly shows a composition that stabilizes the biological activity of TNF. The method consists of combining TNF, also either in solid or liquid form, with a solution containing dissolved albumin. The resulting formulation of TNF is shown to be stable for only short periods of time when subjected to freezing, thawing, or lyophilization. Hereto, the problem associated with TNF oligmer formation is similarly not addressed. Moreover, it is important to point out that neither of these references present formulations that are free of particulate matter.